The goal of this research is to understand the molecular and mechanistic basis of G protein activation, G protein-effector interaction, regulation of GTP hydrolysis, and G protein inhibition. We will investigate the conformational changes G? undergoes from the receptor-bound nucleotide-free G protein complex to the GTP- bound G protein state, the conformational changes that mediate subunit dissociation following GTP binding and the key allosteric interactions needed to terminate the active state of G? with its effectors and RGS proteins. Our hypothesis is that the dynamic regions of G? are critical for its interactions with other proteins throughout the G protein cycle, and these regions can be understood by examining their energetics at the single-amino acid level and probed using mutagenesis, biophysical and structural techniques. We will use an iterative loop between crystallographic approaches, EPR/DEER studies, structural modeling, sequence conservation, evolutionary coupling analysis, mutagenesis and functional assays to discern the role of each residue in the allosteric, intramolecular path of the G protein from the nucleotide binding pocket to its distal interaction sites with partner proteins. To explore the local dynamics and environment of specific regions, biochemical and biophysical data will be used in a continuous feedback cycle with the structural and sequence- derived computation efforts to fine-tune our predictions. In addition, evolutionary conservation, co-evolution, and statistical coupling of residue pairs across chordate phylogeny will be employed to discern how those network mechanisms are conserved yet still selective within each G? subtype. In this grant period, we will seek in specific aim 1 to identify the mechanism and sequence of GTP binding and helical domain closing. In specific aim 2, we will investigate how GTP binding leads to the dissociation of G? from R* and G??. Finally, in specific aim 3, we will identify allosteric interactions necessary for GTP hydrolysis and effector interaction. In particular, we will use our innovative, transdisciplinary approaches to understand how the G?i-RGS4 complex differs from the G?t - RGS9 - PDE? complex, where effector interaction is required for GTP hydrolysis in rod photoreceptors. These studies will lead to a more complete picture of the functional role the Switch regions play, give insight into the mechanism of GTP-dependent dissociation of G? from G?? and the receptor, and will probe the allosteric mechanisms required for effector and RGS interactions culminating in GTP hydrolysis. This iterative, integrated structural biology approach aims to capture the magnitude, direction, and dynamics of the conformational changes in G proteins and the allosteric basis of their interaction with partner proteins.